Apoptotic vesicle-mediated senolytics requires mechanical loading

Rationale: Mechanical force plays crucial roles in extracellular vesicle biogenesis, release, composition and activity. However, it is unknown whether mechanical force regulates apoptotic vesicle (apoV) production. Methods: The effects of mechanical unloading on extracellular vesicles of bone marrow were evaluated through morphology, size distribution, yield, and protein mass spectrometry analysis using hindlimb unloading (HU) mouse model. Apoptosis resistance and aging related phenotype were assessed using HU mouse model in vivo and cell microgravity model in vitro. The therapeutic effects of apoVs on HU mouse model were assessed by using microcomputed tomography, histochemical and immunohistochemical, as well as histomorphometry analyses. SiRNA and chemicals were used for gain and loss-of-function assay. Results: In this study, we show that loss of mechanical force led to cellular apoptotic resistance and aging related phenotype, thus reducing the number of apoVs in the circulation due to down-regulated expression of Piezo1 and reduced calcium influx. And systemic infusion of apoVs was able to rescue Piezo1 expression and calcium influx, thereby, rescuing mechanical unloading-induced cellular apoptotic resistance, senescent cell accumulation. Conclusions: This study identified a previously unknown role of mechanical force in maintaining apoptotic homeostasis and eliminating senescent cells. Systemic infusion of mesenchymal stem cell-derived apoVs can effectively rescue apoptotic resistance and eliminate senescent cells in mechanical unloading mice.

(C) Western blot analysis showed that MG upregulated p16 and p21 expression in MSCs.βactin was used as a protein loading control.

Figure S4 .
Figure S4.Characteristics of apoptotic vesicles (apoVs) derived from MSCs.(A) Scheme illustrating the isolation procedure of apoVs from MSCs.(B) Transmission electron microscopy analysis of mouse MSC-derived apoVs.Scale bar, 100 nm.(C) Western blot analysis showed MSC-apoVs were positive for cleaved Caspase-3.(D and E) Nanoparticle tracking analysis ofapoVs by Zetaview showed a mean diameter of 164.6 nm and -36.17 mV membrane potential of these vesicles.The Zetaview was calibrated using standard beads of 100 nm.PBS was used as a negative control.

Figure
Figure.S5.Senolytics and MSC-apoVs rescue apoptosis resistance in unloading mice.(A) Representative immunostaining showed that hindlimb unloading resulted in a reduced apoptotic rate in the bone marrow compared with that in wildtype mice and unloading mice treated with Senolytics and ApoVs.Scale bars, 50 μm.(B and C) Quantification of cleaved caspase 3 and cleaved caspase 8 positive cells, respectively, n = 4.

Figure S6 .
Figure S6.MSC-apoVs rescue osteoporosis and MSC deficiency in mechanical unloading mice.(A) Representative H&E staining showed that hindlimb unloading resulted in a reduced trabecular bone area (yellow circled area) compared to freely moving wildtype mice.MSC-apoV treatment significantly increased the trabecular bone area.Scale bars: 1 mm for upper panel and 100 μm for lower panel.(B and C) After 2 weeks of MSC-apoV infusion, BrdU labeling and colony formation assays showed that the decreased proliferation and population doubling rates were rescued in hindlimb unloading MSCs.n = 3. Scale bar, 100 μm.

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Figure.S7.Microgravity contributes to senescent cell phenotype.(A) CCK8 assay showed that microgravity resulted in a decreased bioactivity compared with that in control group.(B) SA-β-gal staining of senescent cells increased in MG compared to control after 7 days of recovery phase following 24 h of exposure to MG or normal gravity.